Coxiella burnetii, the etiologic agent of Q fever, is an obligate intracellular bacteria that replicates within an apparently unmodified phagolysosome. Among intracellular pathogens the organisms are novel in their location of replication, extreme stability to stress, and persistence in the environment. Bacterial replication is controlled primarily by activated macrophage/monocyte and PMN killing mechanisms stimulated by a cell mediated response, but the exact nature of these mechanisms is undefined. We propose that survival mechanisms are the principle virulence determinants of C. burnetii. We will evaluate four strategies for their contribution in survival. First, we will define the nature and role in survival of a C. burnetii lifecycle. The working hypothesis is that separable, morphological variants represent stages of cell differentiation with specific roles in intracellular and extracellular survival. Based upon earlier studies and data presented in Preliminary Studies, two major variant forms (large cell variants and small cell variants) differentially express proteins that support a model of metabolically most active dividing cells and stationary forms, respectively. Second, we will characterize the requirement for and acquisition systems used to obtain and regulate iron. The working hypothesis is that C. burnetii must accommodate conditions of limiting and high iron levels to survive in the phagolysosome. Data presented in Preliminary Studies demonstrate a C. burnetii ferric uptake regulator (fur) gene and proteins involved in iron acquisition using a ferric uptake regulator titration assay (FURTA). Third, we will characterize the role of anti-oxidant gene products in survival. The working hypothesis is that C. burnetii express enzymes which detoxify oxygen radicals outside of their cytoplasm and respond to oxidative stress by repairing DNA damage caused by oxygen radicals. Data presented in Preliminary Studies allow us to characterize catalase and RecA. We will test the molecular Koch's postulate for the requirement of RecA by creating a transdominant negative mutant through genetic transformation. Finally, we will characterize the role of acid phosphatase in intracellular survival. All specific aims will be facilitated by an ongoing genome sequence project. The working hypothesis is that C. burnetii express acid phosphatase which phosphorylates phagolysosomal proteins and reduces their antibacterial activity.